The major goal of this project is to develop and exploit tractable, preclinical mouse models of malignant melanoma that recapitulate the clinical progression of surgical removal of primary tumors, treatment with adjuvant/neoadjuvant therapy and clinical recurrence at distant sites. We see a translational opportunity to devise a novel strategy to prevent the clinical recurrence of metastatic disease. The models are being designed to incorporate normal immune system function to maintain an appropriate tumor microenvironment and provide superior tumor-host interactions, an approach that is significantly more likely to yield biologically and clinically relevant data. Our focus will be on immunotherapy, which cannot be studied in human PDXs at this point as mice bearing a fully functional humanized immune system remain a future goal. Since metastasis often targets internal organs and the timing of its recurrence may vary greatly, the preclinical model will allow for the non-invasive, long-term monitoring of disease progression within the immunocompetent mouse. To meet these criteria, we have optically labeled melanoma tissue that was never adapted to cell culture and labeled using a high-titer lentivirus encoding a luciferase/GFP fusion reporter protein, developed by this laboratory in collaboration with Dom Esposito at the Protein Expression Laboratory, Advanced Technology Program (Day et al., Pig Cell Melanoma Res 22:283-295, 2009). However, we have noted that even low expression of the xenobiotic reporters GFP and/or luciferase can render growth of labeled metastatic tumors unpredictable in syngeneic mice and severely limit the utility of any immunocompetent preclinical model. We have therefore developed transgenic mice in both FVB/N and C57BL/6 backgrounds that express the Luc/GFP fusion gene away from the trunk where most metastasis is observed (in this case the anterior pituitary through use of a rat growth hormone promoter) (Day et al., PLoS One. 2014 Nov 4;9(11):e109956). These glowing head mice are thus pre-tolerized to both foreign markers, and we note that clearly labeled metastatic lesions arise much more regularly, consistently, and with a stronger signal in the lungs of inoculated, resected host mice. These mice are currently being employed in our ever-improving preclinical models (Day et al., PLoS One, 2014), and are available for purchase from Jackson Labs. The primary goal now is to combine all the tools and reagents we have developed over the last few years to study the mechanisms by which various melanoma subtypes recur at metastatic sites when patients fail targeted and immunotherapeutic drug treatment. This extensive preclinical effort is being performed in full collaboration with Dr. Shyam Sharan and CAPR, and in collaboration with Drs. Nick Restifo (CCR), Jedd Wolchok (MSKCC) and Jennifer Wargo (MD Anderson). We have developed and characterized 4 different melanoma models that vary in terms of oncogenic drivers and initiating carcinogens. A wildtype BRAF/NRAS/NF1 metastatic melanoma model (so-called triple negative) has been developed using our UV-initiated HGF transgenic mouse. Metastatic BRAF mutant melanomas have also been generated and are being fully characterized and incorporated into our studies. Preclinical trials with immune checkpoint inhibitors will be the focus of our work, and studies using both anti-CTLA-4 and, in collaboration with MedImmune, anti-PD-L1) are already underway and providing novel insights into why some melanomas but not others respond to immune checkpoint inhibitors. The responses in our 4 melanoma models to anti-CTLA-4 and anti-PD-L1 seem appropriate to their clinical counterparts. We have written a PRIMER for Cell that reviews optimal use of preclinical cancer models (Day, Merlino, Van Dyke, CELL, 2015). Our data suggest that neoantigens generated by UV exposure and other sources are, at least in part, responsible for the responses of melanoma tumors to immune checkpoint inhibitors. A number of approaches, including in vivo screens, vaccines, and computational assessment, are underway to identify the neoantigens that are responsible. We are also studying how the immune/inflammatory microenvironment influences the response to immune checkpoint inhibitors, as well as the role of melanoma clonality. Recently, we have developed several highly metastatic melanomas and their derived cell lines. These will be used to study the efficacy of neoadjuvant therapy in immune checkpoint response, which is being introduced in the clinic now. Moreover, we have developed new models for melanoma brain metastasis, which we will be emphasizing as a new subproject as we move forward in the future.